0003- Musarrat Shaheen- Ento. Journal (Jan

Pakistan Entomologist
Journal homepage: www.pakentomol.com
SCREENING AND EVALUATION OF INSECTICIDAL RNAI PARTIAL GENE
CONSTRUCTS IN NON-TARGET INSECT SPECIES
Mussarat Shaheen, Imran Amin and *Shahid Mansoor
Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE),
P O Box 577, Jhang Road, Faisalabad, Pakistan
ARTICLE INFORMATION
Received: November 6, 2013
Received in revised form: December 16, 2013
Accepted: December 20, 2013
*Corresponding Author:
Shahid Mansoor
Email: mishi.imtiaz@gmail.com
A BS TR A C T
Potato Virus X mediated VIGS mechanism was employed for colossal reproduction of
dsRNA in tobacco plants to screen partial gene RNAi constructs of 152bp Calcium channel
alpha 1a (CAα1a, 325bp nuclear gene) and Elongation factor-1a (ELF1-a) from cotton
mealybug P. solenopsis Tinsley and Arginine kinase (AK) from A. gossypii in non-target
insect species. Feeding dsRNA of CAα1a and ELF1-a in aphid species M. persicae Sulzer
exhibited no significant insecticidal RNAi effect in terms of adult aphid mortality and
nymphs reproduction by adult aphids and non-significant insecticidal effects in terms of
larval mortality in H. virscens (p<0.05). Feeding CAα1a showed significant insecticidal
effects on larval mortality and larval development in S. litturalis and dry weight gain in H.
virescens (p<0.05 and p<0.01, respectively). AK exhibited visible and strong significant
insecticidal RNAi effects (p< 0.01) in terms of adult aphid mortality, nymphs production and
survival of M. persicae ,as well as in term of larval mortality and dry weight gain of survived
larvae of S. litturalis and H. virescence on transgenic tobacco plants. The results showed
visible clues of some of the non-target RNAi effects which seems to be highly dependent on
homology and length of nucleotide sequences utilized closest to taxa in evaluation of nontarget RNAi effects.
Keywords: dsRNAs, RNAi, tRNAs, GDP, PVX
Blocking the expression of specific gene targets holds
considerable promise for the development of novel RNAibased insect pest management strategies (Burand and Hunter,
2012). The specificity of RNAi-mediated insecticidal effects
is an important consideration with minimum effects on nontarget insects for the use of RNAi for a practical application in
insect resistant transgenic plant technology (Gatehouse et al.,
2012). Various reports of unintended targeting of genes even
with low level of homology share by target gene sequence
raised the question of RNAi specificity and compromised the
important concern of target specificity even then if these may
not be perfect and in terms of both targeted gene and targeted
organism (Tschuch et al., 2008). Previously, Baum et al.
(2007) tested dsRNAs of β-tubulin, V-ATPase-A and VATPase-E gene from western corn rootworm (WCR),
Diabortica vergifera vergifera, southern corn rootworm
(SCR), Diabrotica undecimpunctata Howardii, colorado
potato beetle (CPB), Leptinotarsa decemlineata and cotton
INTRODUCTION
RNAi is sequence specific highly conserved and promising
RNA based technology of knocking down gene expression in
all eukaryotes including arthropods.(Burand and Hunter,
2012; Gatehouse et al., 2012; Gatehouse, 2008; Zhu et al.,
2012). RNAi is sequence-specific gene silencing mechanism
at post transcription level induced by double stranded RNA
(dsRNA) to disrupt endogenous gene expression and modern
plant biotechnology is offering double stranded RNA
(dsRNA) as an impending insect control strategy to build up
the basis of new generation of insect resistant transgenic crop
plants (Gordon and Waterhouse, 2007; Niu et al., 2010;
Walker and Allen, 2010). Breakthrough reports based on
transgenic RNAi plants expressing dsRNAs and silencing
have signified highly sequence specific functional
universality of RNAi in nematodes and insects (Baum et al.,
2007; Price and Gatehouse, 2008; Dubreuil et al., 2009).
Cite this article as: Shaheen, M., I. Amin and S. Mansoor, 2014. Screening and evaluation of insecticidal RNAi partial gene constructs in non-target insect
species. Pak. Entomol., 36(1):13-20.
13
77
Shaheen et al. / Pakistan Entomologist 2014, 36(1): 13-20
720C 45sec, 720C 10min followed 40C 10min. ELF-1a (NCBI
AB439212) forward primer: ELF-1a 5'
GGTATCGATACAGTACCAGTAGGTAGAGT and ELF1a3' TTTGTCGACCGGGAGTATATCCGTTGGAA as revers
primer and PCR profile: 940C 5min, 940C 3min, 500C
30sec,720C 45sec, 720C 10min followed 40C 10min. Arginine
Kinase (AK) (NCBI GU937512) from A. gossypii gene
specific primer sequence (AK)5'
AAGATCGATACAAAATTTGGATCCACG as forward
primer (AK)3' GAGGTCGACCGTGGAAGAAACCTTATC
as reverse primer and PCR profile: 940C 5min, 940C 3min,
500C 30sec,720C 45sec, 720C 10min followed 40C 10min The
152bp CAα1a,225bp ELF-1a, 402bp AK bp PCR products
were resolved on 1.7% and 2.5% Agrose gel(0.5X TAE
buffer) by gel electrophoresis at 60V using GeneRulerTM
100bp DNA ladder in midigel apparatus (18 x 15cm) and
0.5X TAE (20mM Tris acetate and 0.5mM EDTA [pH 8.0]).
The cloning in PVX was confirmed by respective restriction
endonucleases (Fermentas, Life Sciences) using Cla1and
Sal1 according to compatible buffers and incubation
temperatures for appropriate restriction as recommended by
the manufacturer. The cloned construct of 152bp CA, 225bp
ELF, 402bp AK in PVX binary vector was transformed in
Agrobacterium tumefaciens (GV3101) harboring a helper
pSoup plasmid for binary vector Rep in bacterium by
electroporation using a pulse of 4.8-5.00. Bacterial cultures
were grown at 280C for 48Hours with Kanamycin@ 25μg/ml,
Rifamycin@25μg/ml and Tetracyclin@12.5μg/ml in Luria
Bertani (LB) liquid medium on shaker 220 rpm for growth up
to OD 1. The inoculum were prepared by pelleting the
bacterial cells and suspending the pellet in 10mM MgCl2
adding an equal volume in µl of acetosyringone (final
concentration 100μM) and incubated overnight at 40C. The
construct of 152bp CAα1a/PVX, 325bp ELF1-a/PVX, and
402bp AK/PVX were separately Agro infiltrated in 4-6 week
old tobacco plants which were inoculated and bioassayed
after 12-16 days of post inoculation (dpi) on arrival of mild
PVX symptoms on inoculated leaves. Symptomatic leaves
after 12-16 days after Agro-infiltration expressing 152bp
CAα1a/PVX, 325bp ELF1-a/PVX, 402bp AK through PVX,
empty PVX vector and healthy were bioassayed
independently in completely randomized design (CRD) with
three treatments and 10 numbers of repeats both in vivo and
detached leaf assay. Twenty five (25) adult potato peach
aphids, Myzus persicae Sulzer and 5 neonate larvae of S.
litturalis and H. virescence were released on plants clip caged
for leave while detached leaf assays were done by using
petriplate to encage and tight through film. About 6-8 week
older healthy plants with ample biomass were used in
bioassays. Tobacco plants, N. benthamiana L. used in the
study were raised in glass chamber at 22-250 C and 70-75%
relative humidity. About 4-6 weeks old plants were used for
Agrobacterium-PVX co-infiltration in replicated experiment.
Eggs and neonates larvae of H. virescence and S. litturalis for
control experiments were acquired from Nuclear Institute for
Agriculture and Biology (NIAB), Faisalabad and University
College of Agriculture, Department of Entomology,
Bahauddin Zakariya University (BZU), Multan, Pakistan.
The aphid species of M. persicae was acquired from Aphid
Rearing Laboratory, Boyace Thompson Institute (BTI)
Ithaca, USA. Tested insects were obtained from insect
boll weevil, Anthonomus grandis Boheman with sequence
homology of 83% and 79% induced an effective RNAi
response in terms of significant larval mortality in non-target
insects species cited above. Whereas, A. grandis larvae
showed insensitivity to orthologous dsRNA mediated RNAi
response in terms of larval mortality and phenotypic defects
compared to control (Baum et al., 2007). The transgenic
tobacco plants expressing nuclear receptor complex (ECR)
dsRNA (HaEcR) of Helicoverpa armigera with 89%
homology with Spodoptera exigua were bioassayed for RNAi
response in tested insect causing significant molting defects,
failure to complete pupation, adult emergence and 40% more
lethality compared to control plants (Zhu et al., 2012). A
nucleotide sequence of 480bp dsRNA with 41.6% orthologe
homology in mosquito was failed to induce RNAi response in
terms of phenotypic or mortality but dsRNA were recovered
from mosquito gut (Coy et al., 2012). The orally ingested
species specific dsRNA of V-ATPase A and E gene were
designed across taxa, tested against fruitfly, Drosophila
melanogaster; pea aphids, Acyrthosiphon pisum; tobacco
hornworm, Manduca sexta; and red flour beetle, Tribolium
castaneum and exhibited obvious deleterious effects on
tested insect species but these (Whyard et al., 2009).
Mohamed and Kim (2011) reported little adverse effects of
Integrin β1subunit dsRNA specific to S. exigua with 71%
sequence homology on diamondback moth, Plutella
xylostella and bPx1 expression Integrin β1subunit orthologe
gene caused larval development.
Potato Virus X (PVX) has been extensively used for transient
gene expression and VIGS based gene silencing studies in
plants (Lacomme and Chapman, 2005; Vleeshouwers et al.,
2006). Insect lack RNA dependent RNA polymerase enzyme
(RdRp), therefor, PVX mediated dsRNA production has been
employed in this study for VIGS mediated enormous, cheap
and simple way of reproduction and amplification of dsRNA
in tobacco plants for higher concentrations of dsRNA
available for oral ingestion of insects. Keeping in view the
importance of emerging RNAi technology in insect pest
control, the objectives of this study was to elucidate the target
specificity of insecticidal RNAi gene constructs through
feeding bioassays on non-target insects.
MATERIALS AND METHOD
Extraction of insect nucleotide sequences from database
Nucleotide sequences for various possible target genes were
retrieved by using NCBI, Genebank and Basic Local
Alignment Tool (BLAST) using www.ncbi.nlm.nlh.gov to
analyze the nucleotide homology of the partial gene
sequences used in this study. Two partial gene sequences as
152bp EST of Calcium channel (CAα1a), 325bp, Elongation
Factor (ELF-1a) genes from cotton mealybug Phenacoccus
solenopsis Tinsley and 402bp, Arginine kinase (AK) from
Aphis gossypii Glover were used in this study and cloned in
Potato Virus X based binary vector pgR107 vector. 152bp
ESTs cloned from CAα1a from P. solenopsis cDNA library.
CAα1a5' TCCAATCGAT TGGCCAAGAAAACGTTGAGC
a s
f o r w a r d
p r i m e r :
C A α 1 a 3 '
ACCGGTCGACATTGGAATGAAGTAATGTAT as reverse
primer and PCR profile: 940C 5min, 940C 3min, 500C 30sec,
14
Shaheen et al. / Pakistan Entomologist 2014, 36(1): 13-20
(AK) through PVX by M. persicae caused significant
mortality in terms of mean alive adult aphids/leaf on AK/PVX
(7.6), PVX (21.7) and Healthy (22.2) plants. Nymphs
survived on AK/PVX, PVX and Healthy were 16.8, 51.7 and
51.9 nymphs for 5 days feeding duration at p=0.01% and
p=0.05% (Table 1). The results indicated a highly significant
feeding effect of dsRNA of AK on M. persicae aphid
population, vitality and survival. The linear trend of adult
aphids and nymphs production on AK/PVX plants compared
to control plants was established (Figure 6, (1 & 2).
rearing facilities at National Institute for Biotechnology and
Genetic Engineering (NIBGE). Protocols have been well
established for Agrobacterium infiltration and Agrobacterium
infections to achieve transcription and translation of
transgenes in plants. Virus induced Gene Silencing (VIGS)
mediated dsRNA transient expression through PVX in
tobacco plants was confirmed by Reverse transcriptase (RTPCR) from inoculated plants. RT-PCR was performed to
check PVX based transient expression of partial gene
transcripts of 152bp of CAα1a, 325bp ELF1-a, 402bp (AK)
and along 798bp CPP gene of PVX as control fragment, PVX
empty vector, 798bp CPP gene of PVX, Healthy plants and
+ive control plasmid of ELF-1a and -ive control. Total RNA
from infected plant was extracted by grinding 100 mg
symptomatic leaf of inoculated plants which were collected
and immediately dipped in liquid nitrogen. Total RNA was
extracted by TRI-REAGENT (Invitrogen /Molecular
Research Centre, U.S.A). For preparation of first strand
complementary DNA, the Revert-Aid® H Minus first strand
cDNA synthesis kit (Fermentas, Life Sciences) was used
according to prescribed protocol by using oilgo(dT)primers.
Feeding effect of dsRNA of 152bp of CAα1a and 325bp of
ELF-1a partial gene transcript from P. solenopsis and
402bp AK from A. gossypii on S. litturalis and H.
virescens
Feeding CAα1a a dsRNA on transgenic tobacco plants caused
very few larval mortality and less leaf area damage as well as
stunted larval growth of the tested insects on CAα1a/PVX
plants. Feeding of S. litturalis and H. virescens on
CAα1a/PVX plants imposed some negative effects on larval
survival, growth and development compared to control plants
(Figure 1, Panel 2). Mean alive larvae of S. litturalis on
CAα1a/PVX (4.5), PVX (4.8) and healthy (5.0) and mean dry
weight of its survived larvae on CAα1a/PVX (0.125), PVX
(0.795) and healthy (0.792) plants for 5 days feeding duration
was significant at p=0.05% and p=0.01% (Table 5.2).
Whereas, mean alive larvae of H. virescence on CAα1a/PVX
(4.5), PVX (5.0) and healthy (4.9) plants for 5 days feeding
duration (Figure 1, Panel 3) was non-significant but dry
weight of its survives larvae was significantly lower
(CAα1a/PVX=0.368, PVX=0.703, Healthy=0.766) for 5
days feeding duration at p = 0.01% (Table 1). The linear trend
of larvae survival and dry weight gain of S. litturalis and H.
virescence on CAα1a/PVX compared to control plants was
observed (Figure 2, (3 & 4).
The visible feeding effects of ELF-1a on S. litturalis were as
followed; pale and yellowish color of feeing larvae, stunted
larval growth, reduced/tapering body, less dry weight gain on
ELF-1a/PVX plants as compared to control (Figure 3, Panel
B). Although, mean S. litturalis alive larvae on ELF-1a/PVX
(4.7), PVX (4.8) and healthy (4.9) plants was non significant
but mean of dry weight gain by survived larvae on ELF1a/PVX (0.298), PVX (0.788) and healthy (0.809) plants for 5
days feeding duration was significant at p=0.01% (Table 1). In
case of H. virescence, neonate larvae feeding on ELF-1a/PVX
plants although survived but poorly grown and developed
with reduced larval size and less leaf area damage compared
to control plant ((Figure 3, Panel C). The mean larvae
survived on ELF-1a/PVX (4.4), PVX (5.0) and healthy (4.7)
plants and dry weight gain on ELF-1a/PVX (0.378), PVX
(0.738) and healthy (0.81) plants for 5 days feeding duration
were significant at p=0.05% and p=0.01% respectively (Table
1). The linear trend of larvae survival and dry weight gain of S.
litturalis and H. virescence on ELF-1a /PVX compared to
control plants was observed (Figure 4, (3 & 4).
Feeding of dsRNA of AK caused high larval mortality, very
lower dry weight gain of surviving larvae and less leaf area
damage (Figure 5, Panel B). Mean numbers of alive larvae of
S. litturalis (AK/PVX=1.4, PVX=4.7, healthy=4.8) and dry
weight gain (AK/PVX=0.298, PVX=0.788, healthy=0.809)
RESULTS
Feeding effect of dsRNA of 152bp partial gene transcript
of CAα1a and ELF-1a from P. solenopsis and 402bp AK
from A. gossypii on adult M. persicae population and
survival
The adult aphids normally which were fed on tobacco plants
expressing dsRNA of 152bp CAα1a through PVX survived
and reproduced enormous nymphs/babies on all treated
plants. Moreover, no phenotypic and behavioral change like
feeding deterrence or reproductive abnormality was observed
on CAα1a/PVX and control plants. The adult aphid
profoundly produced nymphs and newborn nymphs also
survived and colonized the leave of plants (Figure 1, Panel A).
Non significant effects had been evaluated for adult aphid
population as well as production and survival of nymph
against p=0.05% (Table 1). The linear trend of adult aphids
survival and nymphs production on CAα1a/PVX was
established compared to control treatments (Figure 2, (1& 2),
respectively.
Tobacco plants expressing 325bp dsRNA for ELF-1a through
PVX explained high adult aphid mortality and less number of
nymphs production on ELF-1A/PVX plants compared to
control plants (Figure 3, Panel A). A significant reduction was
.scored in adult aphid population in terms of aphid mortality
and nymphs production by adult aphids on ELF-1a/PVX
(13.7), PVX (20.9) and Healthy (22.1) as well as in nymph
production on ELF-1a /PVX (24.8), PVX (51.7) and healthy
(54.9) plants at p=0.01% (Table 1). The linear trend of adult
aphids survival and nymphs production on ELF-1A/PVX
plants compared to control plants was established (Figure 4,
(1 & 2).
Significant adult aphid mortality and reduced nymphal
production were observed as most of the adult aphids did not
survive to reproduce, few adult aphids developed into winged
isoforms while neonate nymphs died as soon as they started
feeding (Figure 5, Panel A). Feeding of tobacco plants
expressing 402bp dsRNA partial gene transcript of A. gossypii
15
Shaheen et al. / Pakistan Entomologist 2014, 36(1): 13-20
Table 1
ANOVA for feeding effect of dsRNA 152bp Calcium channel alpha 1a (CAα1a), 325bp Elongation Factor (ELF-1a), partial
gene transcript P. solenopsis Tinsley & 402bp Arginine kinase (AK) partial gene transcript A. gossypii Glover on M. persicae
Sulzer aphid population survival, larval mortality and dry weight of bollworms S. litturalis and H. virescence.
Insect species
Mean
SOV
df
MS
p-value
Adult aphid
M. persicae Sulzer
CAα1a/PVX
PVX
21.4
21.6
Rep
Treats
9
2
2.004NS
0.233NS
0.1157
-
Nymphs
M. persicae Sulzer
Healthy
CAα1a/PVX
PVX
21.3
15.2
17.00
Error
Rep
Treats
18
9
2
1.048
NS
21.259
NS
8.40
0.0030
0.3603
Healthy
CAα1a/PVX
15.8
4.5
Error
Rep
18
9
7.770
0.152
-
PVX
4.8
Treats
2
0.633*
0.0325
Healthy
5.0
Error
18
0.009
-
Dry weight
S. littoralis
CAα1a/PVX
0.125
Rep
9
0.006
-
PVX
Healthy
Larval mortality
H. virescens
CAα1a/PVX
PVX
Healthy
0.795
0.792
4.5
5.0
4.9
Treats
Error
Rep
Treats
Error
2
18
9
2
18
1.49**1
0.007
0.163
0.700NS
0.219
0.000
0.0645
-
Dry weight
H. virescens
CAα1a/PVX
0.368
Rep
9
0.005
-
PVX
Healthy
ELF-1a/PVX
PVX
0.703
0.766
13.70
20.90
Treats
Error
Rep
Treats
2
18
9
2
0.458**
0.009
6.596NS
206.40**
0.0000
0.1295
0.0000
Healthy
22.10
Error
18
3.585
-
ELF-1a /PVX
24.80
Rep
9
321.719
PVX
Healthy
ELF-1a /PVX
51.70
54.90
4.7
Treats
Error
Rep
2
18
9
PVX
4.8
Treats
Healthy
4.9
ELF-1a /PVX
0.298
PVX
Larval mortality
S. littoralis
Adult aphid
M. persicae Sulzer
Nymphs
M. persicae Sulzer
Larval mortality
S. littoralis
Dry weight
S. littoralis
NS
0.1203
2430.10
170.507
0.089
**
0.0002
-
2
0.100
-
Error
18
0.10
-
Rep
9
0.018
-
0.788
Treats
2
0.835
0.0000
-
S.D
S.E
0.467
0.207
5.256
0.960
0.43
0.079
0.330
0.060
0.484
0.088
0.197
0.036
4.302
0.785
19.320
3.527
0.407
0.074
0.276
0.050
0.535
0.098
0.202
0.037
7.106
1.297
Healthy
0.809
Error
18
0.020
Larval mortality
H. virescens
ELF-1a PVX
4.4
Rep
9
0.330
0.1669
PVX
Healthy
5.0
4.7
Treats
Error
2
18
0.900*
0.196
0.0246
-
Dry weight
H. virescens
ELF-1a /PVX
0.378
Rep
9
0.003
-
PVX
Healthy
AK/PVX
0.738
0.810
7.60
Treats
Error
Rep
2
18
9
0.535
0.005
1.870
0.0000
-
PVX
Healthy
AK/PVX
21.70
22.20
16.80
Treats
Error
Rep
2
18
9
687.033**
4.070
254.311 NS
0.0000
0.0243
PVX
Healthy
AK/PVX
PVX
Healthy
AK/PVX
51.70
51.90
1.4
4.7
4.8
0.298
Treats
Error
Rep
Treats
Error
Rep
2
18
9
2
18
9
4083.433*
86.211
0.478NS
37.433**
0.433
0.018
0.0000
0.4084
0.0000
-
20.348
3.715
1.732
0.316
PVX
Healthy
AK/PVX
PVX
Healthy
AK/PVX
0.788
0.809
2.4
4.9
4.8
0.206
Treats
Error
Rep
Treats
Error
Rep
2
18
9
2
18
9
0.835**
0.020
1.144NS
20.033**
0.922
0.005
0.0000
0.3315
0.0000
-
0.276
0.050
1.520
0.277
PVX
0.783
Treats
2
1.073
0.0000
0.287
0.052
Healthy
0.762
Error
18
0.011
-
Adult aphid
M. persicae Sulzer
Nymphs
M. persicae Sulzer
Larval mortality
S. littoralis
Dry weight
S. littoralis
Larval mortality
H. virescens
Dry weight
H. virescens
NS
SOV=Source of variation, Rep= Replication, Treat= Treatments df=degree of freedom, MS= Mean squares, p-value= estimated at corresponding degree of
freedom, df= degree of freedom, S.D=Standard deviation, S.E=Standard error, NS= non significance, *= significance at p=0.05%, **= Significance at p=0.01%
16
Shaheen et al. / Pakistan Entomologist 2014, 36(1): 13-20
Table 2
BLAST results of 152bp Calcium channel alpha 1a (CAα1a), 325bp Elongation Factor (ELF-1a), partial gene transcript P.
solenopsis Tinsley & 402bp Arginine kinase (AK) partial gene transcript A. gossypii Glover with other economically important
insect species.
152bp Calcium channel alpha1a (CAα1a) partial gene transcript P. solenopsis Tinsley
Accession No.
Description
Max
score
Total
score
Query
coverage
E
value
Max
indent
XM001943894.2
PREDICTED: Acyrthosiphon pisum mRNA voltagedependent calcium channel type A subunit alpha-1-like
(LOC100164406),
Aedes aegypti voltage-dependent p/q type calcium channel
partial mRNA
292
292
100%
4e-76
100%
100
100
96%
2e-18
75%
XM003698860.1
PREDICTED: Apis florea voltage-dependent calcium channel
type A subunit alpha-1-like (LOC100864653), mRNA
123
123
100%
3e-25
80%
XM003394356.1
PREDICTED: Bombus terrestris voltage-dependent calcium
channel type A subunit alpha-1-like (LOC100647135), mRNA
113
113
100%
5e-22
79%
NM001165904.1
Apis mellifera cacophony (Cac), mRNA, [GQ202019.1] Apis
mellifera cacophony (CAC) mRNA, complete cds
114
114
100%
1e-22
78%
NM001272548.1
Drosophila melanogaster cacophony (cac), transcript variant
P, mRNA
95.1
95.1
98%
1e-16
73%
XM001943894.2
PREDICTED:Acyrthosiphon pisum voltage-dependent
calcium channel type A subunit alpha-1-like
(LOC100164406), mRNA
82.4
82.4
96%
6e-13
72
NM001165910.1
Tribolium castaneum cacophony B (CAC) mRNA, complete
cds
69.8
69.8
96%
4e-09
71%
GQ202018.1
Bombyx mori cacophony (CAC) mRNA, partial cds
68.0
68.0
96%
1e-08
70%
XM312358.4
325bp Elongation Factor 1a (ELF-1a) partial gene transcript P. solenopsis Tinsley
AB439212.1
Phenacoccus sp. PAK EF-1a gene for elongation factor 1a,
partial cds
470
579
100%
3e129
100%
AY427241.1
Phenacoccus solani elongation factor 1a (EF-1a) gene, partial
cds
452
561
100%
8e124
100%
FJ768770.1
H. virescence elongation factor 1alpha(EF-1a) gene partial cd
208
208
65%
2e-54
77%
AF151624.1
Spodoptera litturalis elongation factor-1 alpha (EF-1 alpha)
gene, partial cds
212
212
65%
2e-56
78%
EF419315.1
Myzus persicae isolate Fuzhou population elongation factor 1
alpha (EF1a) gene, partial cds
208
208
98%
9e-56
71%
402bp Arginine kinase (AK) partial gene transcript A. gossypii Glover
GU797831.1
Aphis gossypii arginine kinase-like mRNA, complete sequence
618
618
100%
1e-173
100%
XR045869.2
Predicted: Acyrthosiphon pisum mRNA arginine kinase-like
(LOC100166732),
464
464
100%
1e-127
90%
GU726905.1
Helicoverpa. virescens arginine kinase mRNA, complete cds
165
165
97%
3e 37
70%
HQ840714.
Spodpterea litura arginine kinase (AK) mRNA, complete cds
140
140
92%
4e-35
69%
HQ327310.1
Plutella xylostella arginine kinase mRNA, complete cds
125
125
97%
2e-25
67%
for 5 days feeding duration was estimated significant at
p=0.01% (Table 1). Feeding effect of dsRNA of AK by H.
virescence suffered high larval mortality, very lower dry
weight gain and a less leaf area damage (Figure 5, Panel C).
Alive larvae on AK/PVX (2.4), PVX (4.9) and healthy (4.8)
plants and mean dry weight of survived larvae of H.
virescence on AK/PVX (0.206), PVX (0.783) and healthy
(0.762) for 5 days feeding duration were significant at
p=0.01% (Table 1). The linear trend of larvae survival and dry
weight gain of S. litturalis and H. virescence on AK/PVX
compared to control plants were recorded (Figure 4, (3 & 4).
DISCUSSION
Three highly conserved housekeeping genes have been
utilized to determine the specificity of RNAi. Voltage gated
calcium channels are central to calcium-dependent gene
transcription, muscle contraction, cardiac action potential
propagation and CaV regulation and plays a vital role in
endocrine by hormone neurotransmitter release (Catterall,
2011; Minor and Findeisen, 2010). Elongation factor 1a (EF1a) in all organisms including insect species is a nuclear protein
coding. Gene is part of an enzymatic complex involved in the
17
Shaheen et al. / Pakistan Entomologist 2014, 36(1): 13-20
Panel A
Panel A
Panel B
Panel B
Panel C
Panel C
Fig. 1
Feeding effect of dsRNA 152bp partial gene transcript CAα1a
of P. solenopsis expressed in tobacco plants through PVX
vector on M. persicae adult aphids and nymphs (Panel A), S.
litturalis ( Panel B) and H. virescence (Panel C) larvae,
respectively survival and development compared to control
plants, where(A)= CAα1a/PVX, (B)=PVX and (C)=Healthy
as control plants.
Fig. 3
Feeding Effect of dsRNA 325bp partial gene transcript ELF1a of P. solenopsis expressed in tobacco plants through PVX
vector on M. persicae adult aphids and nymphs (Panel A), S.
litturalis (Panel B) and H. virescence larvae (Panel C),
respectively survival and development compared to control
plants, where(A)= ELF-1a /PVX, (B)=PVX & (C)=Healthy
as control plants.
Fig. 2
Feeding effect of dsRNA 152bp partial gene transcript CAα1a
cotton mealybug P. solenopsis on (1)= population survival of
adult M. persicae, (2)= Number of nymphs produced (3)= S.
litturalis (4)= H. virescence respectively larvae survival and
development compared to control plants.
Fig. 4
Feeding effect of dsRNA 325bp partial gene transcript ELF 1a
cotton mealybug P. solenopsis on (1)= population survival of
adult M. persicae, (2)= Number of nymphs produced (3)= S.
litturalis (4)= H. virescence respectively larvae survival and
development compared to control plants.
GTP dependent binding of charged aminoacyl-transfer RNAs
(tRNAs) relocate to the acceptor site (A site) of the 80S
ribosomal subunits during translation (Stuart and
Chamberlain, 2003; Zhou et al., 2008). Arginine kinase (AK)
is a phospho-transferase enzyme that executes a critical role in
cellular energy metabolism in invertebrates (Zhou et al.,
2008). Genes encoding proteins with crucial biological
functions are the best RNAi target genes as they might cause
mortality/lethal effects, clearly morose growth and
development and phenotypic abnormalities (Mito et al.,
2010). Hence, parameters understudy are appropriate
indicator of RNAi effects in insects. In answer to the question
whether other insect species will be affected by the
application of non-specific dsRNA in non-target insect
species, the results of CAα1a nucleotide sequence from
cotton mealybug P. solenopsis was BLAST against other
insect species to access sequence homology for establishing
analogy in statistical and molecular results of these bioassays.
The BLAST results of 152bp CAα1a partial gene transcript
from P. solenopsis revealed 80% similarity with A. pisum
(Table 1) and BLAST did not resulted any orthologe found
from M. persicae although estimated 14.4% mortality in adult
M. persicae and 2.3% less nymphs were produced compared
to control. The BLAST results revealed H. virescence and S.
18
Shaheen et al. / Pakistan Entomologist 2014, 36(1): 13-20
Panel A
Panel B
Panel C
Fig. 5
Feeding Effect of dsRNA 402bp partial gene transcript (AK)
of A. gossypii Glover expressed in tobacco plants through
PVX vector on (Panel A)= M. persicae Sulzer adult aphids&
nymphs,( Panel B)= S. litturalis, (Panel C)= H. virescence
larvae respectively survival and development compared to
control plants, where(A)= AK /PVX, (B)=PVX &
(C)=Healthy as control plants.
Fig. 6
Feeding effect of dsRNA 325bp partial gene transcript AK of
A. gossypii Glover on (1)= population survival of adult M.
persicae Suzler, (2)= Number of nymphs produced (3)= S.
litturalis (4)= H. virescence respectively larvae survival and
development compared to control plants.
litturalis nucleotide homology (˜10% mortality) was nonsignificant but dry weight of survived larvae was significantly
lower than larvae fed on controls. The results indicated that
feeding of non-specific dsRNA of CAα1a on non-target insect
species reflected its non-significant effects on insect mortality
was but significant effects on development in terms of dry
weight gain for H. virescence and S. litturali larvae.
Previously RNAi effects scored gene, 20 hydroxyecdysone
receptor complex in S. exigua were as deleterious as revealed
in this study although, sequence homology was 89% with H.
virescence ECR gene reported by Mohamed and Kim (2011)
and Zhu et al., 2012. The results of present studies could help
to predict and evaluate the commonly harmful gene knocking
down effects of feeding dsRNA of CAα1a with crucial
biological function.
A phenotype resulting from knockdown of gene expression
may also enlighten the target gene function and certainly
helpful to characterize target gene effects (Alves et al., 2011).
In this study, ELF-1a of cotton mealybug P. solenopsis
revealed 12% and 6% more mortality of H. virescence and S.
litturalis, respectively compared to control while BLAST
results of orthologe sequence homology was 77% and 78%,
respectively. The dsRNA significantly affected the
development of larvae in term of significant reduction in dry
weights of the larvae but ELF-1a of cotton mealybug P.
solenopsis caused a significant mortality explaining 45.2%
more adult mortalityand 52% less nymphs compared to
control (Table 1) and nucleotide sequence homology was
71% of M. persicae (Table 2). The AK gene is highly
conserved in insects and AK from cotton aphid A. gossypii, by
BLAST generated 90% of sequence homology with A. pisum
but this insect is not tested in this study. The bioassay H.
virescence and S. litturalis with 70% and 69% sequence
homology in BLAST (Table 2) revealed a 52% and 72% more
mortality in respective insects compared to control and
statistical results were highly significant for mortality and dry
weight gain of surviving insects while none of the Arginine
kinase orthologous could be found in database for M.
persicae. But results of bioassay revealed an estimated 69.6%
more adult mortality than control. Arginine kinase feeding
dsRNA exerted a drastic effect on Myzus aphid mortality and
survival with 30% lesser number of nymphs production by M.
persicae compared to controls. The BLAST results discussed
helped in concluding that nucleotide homology of partial gene
transcript could help us to explain the significant mortality
effects for the insects of same order.
RNAi effects are highly dependent on the amount of dsRNA
intake by the tested organism and perfect sequence homology
of designed dsRNA that have been employed (Tschuch et al.,
2008). To evaluate the silencing efficiency of RNAi, method
of delivery of dsRNA has to be taken into account It is
generally assumed that RNAi will always occur once dsRNA
is delivered inside the insect cell and limiting factor subsists at
the level of its functional uptake, nucleotide homology of
dsRNA with target gene, concentration of dsRNA up take,
type of the gene targeted and presence of systemic RNAi
machinery (Terenius et al., 2011). There is a great variation in
sensitivity, strength and severity of the effects of RNAi for
homologous gene transcripts in various insect species
(Garbutt and Reynolds, 2012). Three partial gene transcripts
of 152bp, 325bp and 402bp have been utilized to express long
dsRNA of corresponding gene transcript in tobacco plant
through PVX and also vet that the effective length of dsRNA
>300bp was effective to induce RNAi in non-target insects.
The dsRNA of 152bp was scored to be ineffective to produce
significant insecticidal effects in non-target insects so, a long
chain of >300bp nucleotides of dsRNA might be effective to
establish coincidental homology/consecutive conserved
region to induce effective RNAi response in non-target insect
species. The dsRNA 152bp of CAα1a partial gene transcript
19
Shaheen et al. / Pakistan Entomologist 2014, 36(1): 13-20
Lacomme, C. and S. Chapman, 2005. Use of Potato Virus X
(PVX)–Based Vectors for Gene Expression and Virus-Induced
Gene Silencing (VIGS). In "Current Protocols in Microbiology".
John Wiley & Sons, Inc.
Minor, D.L., Jr. and F. Findeisen, 2010. Progress in the structural
understanding of voltage-gated calcium channel (CaV) function
and modulation. Channels (Austin)., 4(6): 459-74.
Mito, T., T. Nakamura, T. Bando, H. Ohuchi and S. Noji, 2010. The
advent of RNA interference in entomology. Entomol. Sci., 14(1):
1-8.
Mohamed, A.A.M. and Y. Kim, 2011. A target-specific feeding
toxicity of β1 integrin dsRNA against diamondback moth,
Plutella xylostella. Archives. Ins. Biochem. Physiol., 78(4): 216230.
Niu, J.H., H. Jian, J.M. Xu, Y.D. Guo and Q. Liu, 2010. RNAi
technology extends its reach: Engineering plant resistance against
harmful eukaryotes. Afr. J. Biotech., 9(45): 7573-7582.
Price, D. and J. Gatehouse, 2008. RNAi-mediated crop protection
against insects. Trends Biotech., 26(7): 393 - 400.
Stuart, M.K. and N.R. Chamberlain, 2003. Monoclonal antibodies to
elongation factor-1α inhibit in vitro translation in lysates of Sf21
cells. Archives. Ins. Biochem. Physiol., 52(1): 17-34.
Terenius, O., A. Papanicolaou, J.S. Garbutt, I. Eleftherianos, H.
Huvenne, S. Kanginakudru, M. Albrechtsen, C. An, J.L. Aymeric,
A. Barthel, P. Bebas, K. Bitra, A. Bravo, F. Chevalier, D.P.
Collinge, C.M. Crava, R.A. de Maagd, B. Duvic, M. Erlandson, I.
Faye, G. Felföldi, H. Fujiwara, R. Futahashi, A.S. Gandhe, H.S.
Gatehouse, L.N. Gatehouse, J.M. Giebultowicz, I. Gómez, C.J.P
Grimmelikhuijzen, A.T. Groot, F. Hauser, D.G. Heckel, D.D.
Hegedus, S. Hrycaj, L. Huang, J.J. Hull, K. Iatrou, M. Iga, M.R.
Kanost, J. Kotwica, C. Li, J. Li, J. Liu, M. Lundmark, S.
Matsumoto, M. Meyering-Vos, P.J. Millichap, A. Monteiro, N.
Mrinal, T. Niimi, D. Nowara, A. Ohnishi, V. Oostra, K. Ozaki, M.
Papakonstantinou, A. Popadic, M.V. Rajam, S. Saenko, R.M.
Simpson, M. Soberón, M.R. Strand, S. Tomita, U. Toprak, P.
Wang, C.W. Wee, S. Whyard, W. Zhang, J. Nagaraju, R.H.
Ffrench-Constant, S. Herrero, K. Gordon, L. Swevers and G.
Smagghe, 2011. RNA interference in Lepidoptera: An overview
of successful and unsuccessful studies and implications for
experimental design. J. Ins. Physiol., 57(2): 231-245.
Tschuch, C., A. Schulz, A. Pscherer, W. Werft, A. Benner, A. HotzWagenblatt, L. Barrionuevo, P. Lichter and D. Mertens, 2008. Offtarget effects of siRNA specific for GFP. Bio. Med. Central Mol.
Biol., 9(1): 60-74.
Vleeshouwers, V.G.A.A., J.D. Driesprong, L.G. Kamphuis, T. TortoAlalibo, K.A.E. Van'T Slot, F. Govers, R.G.F. Visser, E. Jacobsen
and S. Kamoun, 2006. Agroinfection-based high-throughput
screening reveals specific recognition of INF elicitins in Solanum.
Mol. Plant. Pathol., 7(6): 499-510.
Walker, W.B. and M.L. Allen, 2010. RNA interference-mediated
knockdown of IAP in Lygus lineolaris induces mortality in adult
and pre-adult life stages. Entomologia Experi. Applicata, 138(2):
83-92.
Whyard, S., A.D. Singh and S. Wong, 2009. Ingested doublestranded RNAs can act as species-specific insecticides. Ins.
Biochem. Mol. Biol. 39(11): 824-832.
Zhou, X., M.M. Wheeler, F.M. Oi, and M.E. Scharf, 2008. RNA
interference in the termite Reticulitermes flavipes through
ingestion of double-stranded RNA. Ins. Biochem. Mol. Biol.,
38(8): 805-815.
Zhu, J.Q., S. Liu, Y. Ma, J.Q. Zhang, H.S. Qi, Z.J. Wei, Q. Yao, W.Q.
Zhang and S. Li, 2012. Improvement of pest resistance in
transgenic tobacco plants expressing dsRNA of an insectassociated gene EcR. PLoS ONE., 7(6): e38572.
Zhu, K.Y., 2013. RNA interference: A powerful tool in
entomological research and a novel approach for insect pest
management. Ins. Sci., 20(1): 1-3.
in target insect P. solenopsis was highly efficient RNAi gene
to induce >90 mortality and severe phenotypic abnormalities
(unpublished data). This study highlighted the valuable
application of PVX vector for screening of partial gene
transcripts to evaluate non target RNAi effects in non-target
insect species.
This study provided us valuable clue of some of the effects of
non-specific RNAi in somewhat closely related insect taxa.
The inherent specificity of RNAi should be conserved by
designing highly conserved short nucleotide stretches of
dsRNA to minimize the chances of consecutive nucleotide
matches with homologous and orthologous gene copies in
non-target insect species for designing more ecologically
friendly approaches for the control of insect pests. The results
of non-specific dsRNA in non-target insects suggest careful
application of dsRNA based technology with resilient need to
design sequence specific and problem specific dsRNAs and
their screening and evaluation through bioassays on nontarget insect species. Keeping in view the essence of RNAi
specificity we emphasize on screening of potential insect
genes that work specifically in insects to target specific insect
pest problems for potential application RNAi technology in
insect resistant transgenic plant development.
REFERENCES
Alves, A.P., M.D. Lorenzen, R.W. Beeman, J.E. Foster and B.D.
Siegfried, 2011a. RNA interference as a method for target-site
screening in the western corn rootworm, Diabrotica virgifera
virgifera. J. Ins. Sci., 10(162): 1-16.
Baum, J.A., T. Bogaert, W. Clinton, G.R. Heck, P. Feldmann, O.
Ilagan, S. Johnson, G. Plaetinck, T. Munyikwa, M. Pleau, T.
Vaughn and J. Roberts, 2007. Control of coleopteran insect pests
through RNA interference. Nat. Biotechnol., 25(11): 1322-1326.
Burand, J.P. and W.B. Hunter, 2013. RNAi: Future in insect
management. J. Inverteb. Pathol., 112: 68-74).
Catterall, W.A., 2011. Voltage-gated calcium channels. Cold Spring
Harb. Perspect. Biol., 11.
Coy, M.R., N.D. Sanscrainte, K.C. Chalaire, A. Inberg, I. Maayan, E.
Glick, N. Paldi and J.J. Becnel, 2012. Gene silencing in adult
Aedes aegypti mosquitoes through oral delivery of doublestranded RNA. J. App. Entomol., 136(10): 741-748.
Dubreuil, G., M.P. Magliano, J. Dubrana, P. Lozano, B. Lecomte, P.
Favery, P. Abad and M.N. Rosso, 2009. Tobacco rattle virus
mediates gene silencing in a plant parasitic root-knot nematode. J.
Exper. Bot., 60(14): 4041-4050.
Garbutt, J.S. and S.E. Reynolds, 2012. Induction of RNA
interference genes by double-stranded RNA; implications for
susceptibility to RNA interference. Ins. Biochem. Mol. Biol.,
42(9): 621-628.
Gatehouse, A., N. Ferry, G. Smagghe and I. Diaz, 2012. Successes
and Failures in Plant-Insect Interactions: Is it Possible to Stay One
Step Ahead of the Insects? Arthropod-Plant Interactions, Vol. 14,
pp. 89-126. Springer Netherlands.
Gatehouse, J.A., 2008. Biotechnological Prospects for Engineering
Insect-Resistant Plants. Plant Physiol., 146(3): 881-887.
Gatehouse, J.A., D.R.G. Price and A. Vilcinskas, 2012. protection of
crops against insect pests using RNA Interference. Insect
Biotechnol., 2: 145-168.
Gordon, K.H.J. and P.M. Waterhouse, 2007. RNAi for insect-proof
plants. Nat. Biotech., 25(11):1231-1232.
Kumar, M., G.P. Gupta and M.V. Rajam, 2009. Silencing of
acetylcholinesterase gene of Helicoverpa armigera by siRNA
affects larval growth and its life cycle. J. Ins. Physiol., 55(3): 273278.
20